Differential transcript isoform usage pre- and post-zygotic genome activation in zebrafish

BMC Genomics Differential transcript isoform usage pre- and post-zygotic genome activation in zebrafish Hvard Aanes 0 Olga strup Ingrid S Andersen Lars F Moen 0 Sinnakaruppan Mathavan Philippe Collas Peter Alestrom 0 0 BasAM, Norwegian School of Veterinary Science , 0033 Dep, Oslo , Norway Background: Zebrafish embryos are transcriptionally silent until activation of the zygotic genome during the 10th cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here, we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA). Results: We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3' untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3' terminal exon when transcribed by the zygote compared to their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped exon events reveal a correlation between histone H3K36 trimethylation peaks and skipped exons, suggesting epigenetic marks being part of alternative splicing regulation. Conclusions: The novel isoforms and isoform switches reported here include regulators of transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes. Zebrafish; Mid-blastula Transition; Zygotic Genome Activation; Alternative Splicing; Transcriptional Start Site; 3'UTR - Background During the first ten cell cycles after fertilization, the zebrafish embryo is transcriptionally silent and consists of undifferentiated and rapidly dividing blastomeres [1]. Initiation of transcription at the 10th cell cycle is termed zygotic genome activation (ZGA). At the time of ZGA, development proceeds from a control by mRNAs synthesized during oogenesis and stored in the egg (maternal transcripts) to mRNAs produced by the embryos own genome. Following ZGA, blastomeres divide less frequently and more asynchronously, they start to differentiate and migrate to form the three germ layers of the gastrulating embryo [1-4]. Collectively, these transformations characterize the mid-blastula transition (MBT). These fundamental cellular and functional changes occurring during development make zebrafish an attractive model to study transcriptional changes governing between pre-MBT and post-MBT development. Previous studies have shown essential roles of activation and degradation of maternal transcripts in regulating the MBT and ZGA [5-7]. Signaling pathways involving Bmp, Nodal, Fgf, Wnt and maternal -catenin are essential for the formation of germ layers and body axis [8]. We and others have shown that the establishment of posttranslationally modified histones on specific genomic sites and DNA methylation play a role in transcriptional regulation around the time of ZGA by patterning developmental gene expression [9-12]. However, although several early transcriptomes have recently been published [5,13,14], little is known on the isoform-specific dynamics governing developmental transitions around the MBT. The notion that each gene can give rise to multiple mRNAs has evolved from being reported as a rare phenomenon [15,16] to include virtually all loci in man [17,18], and has been shown to be crucial for differentiation [19], development [20] and human disease [21]. Isoform switches, defined as a change in the isoform composition of gene products between two conditions (e.g. two developmental stages) are of particular interest since such events are critical for differentiation [19]. The differences between transcript isoforms can affect the coding sequence (CDS) and/or untranslated regions (UTRs) 3' or 5' of the CDS. The former is likely to affect protein function, while the latter may affect translational efficiency, mRNA degradation kinetics and spatial distribution of transcripts [22,23]. The mechanisms regulating splicing and determining the production of specific transcript isoforms have started to be unveiled and involve cis-elements and trans-acting factors, as well as epigenetic modifications in the proximity of spliced exons [24-26]. A genome-wide landscape of transcript isoforms synthesized during early development, and particularly of the switches occurring between maternal and zygotic isoforms at the time the embryo initiates its own transcriptional program, has been lacking and has hampered a comprehensive appreciation of the transcriptional dynamics occurring at the time of ZGA. We have used an isoform prediction and quantification program, Cufflinks [27], to detect novel isoforms and quantify isoform-specific changes from RNA-sequencing (RNA-seq) reads before and after ZGA in zebrafish. We identify numerous novel isoforms related to shifts in transcription start site (TSS), alternative splicing (AS) events and transcription termination sites (TTS), when comparing transcripts of maternal and zygotic origin coming from the same gene. These include transcripts of genes involved in cell-cell interactions, pluripotency control and DNA methylation. Using H3K4 and H3K36 trimethylation (me3) data acquired by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we find that H3K4me3 can form relatively broad domains which cannot distinguish between closely spaced alternative TSSs, unless TSSs are linked to alternative promoters at distant locations. Skipped exons are enriched in H3K36me3 and H3K4me3, extending previous reports on the involvement of these histone modifications in the regulation of isoform usage. Results and discussion Mapping of RNA-seq reads achieved from combined use of two aligners Using two different short RNA-seq read aligning programs, namely Bioscope (Life Technologies, USA, Carlsbad, CA) and TopHat [28], we remapped all reads generated by RNA-seq in a recently published study [5]. This two aligner approach yields a higher number of mapped reads than each aligner alone and proves to be complementary compared to using either aligner separately (Figure S1 and S2 in Additional file 1). From our RNA-seq data, four datasets from pre-ZGA zebrafish developmental stages (pre-MBT) and two from post-ZGA stages (post-MBT) were merged, respectively generating one maternal transcriptome d (...truncated)